direct comparison of different stem cell types and subpopulations

Journal of the American College of Cardiology Vol. 59, No. 10, 2012© 2012 by the American College of Cardiology Foundation ISSN 0735-1097/$36.00

PRE-CLINICAL RESEARCH

Direct Comparison of DifferentStem Cell Types and Subpopulations RevealsSuperior Paracrine Potency and MyocardialRepair Efficacy With Cardiosphere-Derived Cells

Tao-Sheng Li, MD, PHD,*† Ke Cheng, PHD,* Konstantinos Malliaras, MD,*Rachel Ruckdeschel Smith, PHD,*‡ Yiqiang Zhang, PHD,* Baiming Sun, MD,*Noriko Matsushita, MD, PHD,* Agnieszka Blusztajn, BS,‡ John Terrovitis, MD,§Hideo Kusuoka, MD, PHD,� Linda Marban, PHD,*‡ Eduardo Marban, MD, PHD*

Los Angeles, California; Nagasaki and Osaka, Japan; and Athens, Greece

Objectives The goal of this study was to conduct a direct head-to-head comparison of different stem cell types in vitro for variousassays of potency and in vivo for functional myocardial repair in the same mouse model of myocardial infarction.

Background Adult stem cells of diverse origins (e.g., bone marrow, fat, heart) and antigenic identity have been studied forrepair of the damaged heart, but the relative utility of the various cell types remains unclear.

Methods Human cardiosphere-derived cells (CDCs), bone marrow–derived mesenchymal stem cells, adipose tissue–derived mesenchymal stem cells, and bone marrow mononuclear cells were compared.

Results CDCs revealed a distinctive phenotype with uniform expression of CD105, partial expression of c-kit and CD90,and negligible expression of hematopoietic markers. In vitro, CDCs showed the greatest myogenic differentiationpotency, highest angiogenic potential, and relatively high production of various angiogenic and antiapoptotic-secreted factors. In vivo, injection of CDCs into the infarcted mouse hearts resulted in superior improvement ofcardiac function, the highest cell engraftment and myogenic differentiation rates, and the least-abnormal heartmorphology 3 weeks after treatment. CDC-treated hearts also exhibited the lowest number of apoptotic cells.The c-kit� subpopulation purified from CDCs produced lower levels of paracrine factors and inferior functionalbenefit when compared with unsorted CDCs. To validate the comparison of cells from various human donors,selected results were confirmed in cells of different types derived from individual rats.

Conclusions CDCs exhibited a balanced profile of paracrine factor production and, among various comparator cell types/sub-populations, provided the greatest functional benefit in experimental myocardial infarction. (J Am Coll Cardiol2012;59:942–53) © 2012 by the American College of Cardiology Foundation

Published by Elsevier Inc. doi:10.1016/j.jacc.2011.11.029

It is now recognized that resident cardiac stem cells exist inthe adult mammalian heart, including mouse, rat, and

From *Cedars-Sinai Heart Institute, Los Angeles, California; †Department of StemCell Biology, Nagasaki University Graduate School of Biomedical Science, Nagasaki,Japan; ‡Capricor Inc., Los Angeles, California; §Third Department of Cardiology,University of Athens, Athens, Greece; and the �Osaka National Hospital, Osaka,Japan. This study was supported by the National Institutes of Health (HL095203) toCapricor, Inc., by the National Institutes of Health (U54 HL081028) to Dr. E.Marban, and by the Cedars-Sinai Board of Governors Heart Stem Cell Center. Dr.E. Marban is the Mark S. Siegel Family Professor of the Cedars-Sinai MedicalCenter. Dr. E. Marban and Dr. L. Marban hold founders’ equity in Capricor, Inc.Drs. Smith, Blusztajn, Terrovitis, and L. Marban are employed by Capricor, Inc. Drs.Malliaras and Terrovitis are consultants of Capricor, Inc. All other authors havereported that they have no relationships relevant to the contents of this paper todisclose. Drs. Li, Cheng, and Malliaras contributed equally to this work.

Manuscript received August 24, 2011; revised manuscript received October 19,2011, accepted November 22, 2011.

human (1–7). Several groups have expanded the smallpopulation of cardiac stem cells from adult human hearttissue with a view to clinical applications in regenerativetherapy (7–9). We have developed a technique to expand tens ofmillions of cardiosphere-derived cells (CDCs), a mixture ofcardiac stem cells and supporting cells, from percutaneousendomyocardial biopsies (8). This technology is in Phase Iclinical study for the treatment of post-ischemic heart failure(CADUCEUS [Cardiosphere-Derived Autologous StemCells to Reverse Ventricular Dysfunction; NCT00893360]).Nevertheless, heart-derived cells are relative newcomers toregenerative cardiology.

Multiple extracardiac cell sources, including bone marrowmononuclear cells (BM-MNCs), bone marrow–derived
mesenchymal stem cells (BM-MSCs), adipose tissue–
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943JACC Vol. 59, No. 10, 2012 Li et al.March 6, 2012:942–53 Myocardial Repair by Different Types of Stem Cells

derived mesenchymal stem cells (AD-MSCs), endothelialprogenitor cells, and myoblasts, have been used clinically inattempts to regenerate the damaged heart (10–18). Theimplantation of these cells of extracardiac origin has beenfound to produce generally positive effects, mostly throughparacrine mechanisms (19–22). Although resident cardiacstem cells can mediate direct cardiogenesis and angiogenesis(1–7,23,24), recent studies have found that even these cellsexert most of their benefits via indirect paracrine mechanisms(25,26). Therefore, no convincing evidence supports the supe-riority of heart-derived cells for myocardial repair. Even withinheart-derived cells, 2 cell products are in clinical trials, but thesehave yet to be compared directly. The first, CDCs, are anatural mixture of stromal, mesenchymal, and progenitor cells(8); the second cell product represents the c-kit� subpopulationpurified from mixed heart-derived cells (1,9).

In the current study, we performed a head-to-head compar-ison of different stem cell types/subpopulations for functionalmyocardial repair by assessing multiple in vitro parameters,including secretion of relevant growth factors, and in vivo cellimplantation into an acute myocardial infarction model insevere combined immunodeficiency (SCID) mice.

Methods

Cell sources. Human CDCs were expanded as previouslydescribed from minimally invasive endomyocardial biopsies(27). Human BM-MSCs and BM-MNCs were purchasedfrom Lonza (Walkersville, Maryland). Human AD-MSCswere purchased from Invitrogen (Carlsbad, California).These cells were freshly isolated from healthy donors. Thec-kit� stem cell subpopulation was purified from CDCssing a CELLection Pan Mouse IgG Kit and a DynalPC-15 magnetic particle concentrator (Invitrogen).For confirmatory rat studies, 4-month-old Wistar Kyoto

ats were used to expand CDCs, BM-MSCs, and AD-SCs, as described previously (23,28,29). BM-MNCs

ere also collected from the same rats by using gradiententrifugation (19). Freshly collected BM-MNCs andwice-passaged CDCs, BM-MSCs, and AD-MSCs weresed for rat experiments.Unless otherwise noted, basic Iscove’s modified Dulbec-

o’s medium (IMDM) (Invitrogen) supplemented with 10%etal bovine serum (Thermo Scientific HyClone) and gen-amycin 20 mg/ml were used to culture all cell lines.low cytometry. The identity of CDCs, BM-MSCs,D-MSCs, and BM-MNCs was investigated using flow

ytometry, as described previously (6,8). Briefly, cells werencubated with fluorescein isothiocyanate 7– or phycoerythrin-onjugated antibodies against CD29, CD31, CD34, CD45,D90, CD105, CD117 (c-kit), and CD133 (eBioscience,

nc., San Diego, California) for 30 min. Isotype-identicalntibodies served as negative controls. Quantitative anal-sis was performed using a CYAN-ADP flow cytometerith Summit 4.3 software (Beckman Coulter, Brea,
alifornia) (6,8). E
nzyme-linked immunoadsorbentssay. To compare the potency ofhe production of growth factors,ells were seeded in 24-well culturelates at densities of 1 � 106/mlBM-MNCs) or 1 � 105/ml (all

other cell types) in fetal bovine se-rum–free IMDM media (all celltypes) for 3 days. The supernatantswere collected, and the concentra-tions of angiopoietin-2, basic fibro-blast growth factor (bFGF), hepato-cyte growth factor (HGF),insulin-like growth factor (IGF)-1,platelet-derived growth factor, stro-mal cell–derived factor (SDF)-1,and vascular endothelial growth fac-tor (VEGF) were measured withhuman enzyme-linked immunoad-sorbent assay (ELISA) kits (R&DSystems Inc., Minneapolis, Minne-sota), according to the manufactur-er’s instructions. Given the limitednumber of rat-specific ELISA kits,we only measured the concentra-tions of HGF (B-Bridge Interna-tional, Inc., Cupertino, California),IGF-1, and VEGF in the superna-tant with 3 days’ culture of rat cells(R&D Systems Inc.).

To compare the production ofgrowth factors from the purifiedc-kit� subpopulation and unsortedCDCs, we seeded cells (5 � 104/ml)on 24-well culture plates and culturefor 2 days under 20% O2. Growthactors in conditioned media wereeasured by using ELISA as de-

cribed earlier.mmunostaining. To determine myogenic differentiationn vitro, cells were seeded on fibronectin-coated, 4-chamberulture slides. After 7 days of culture, cells were fixed,locked with goat serum for 30 min, and then incubatedith mouse anti-human troponin T antibody (R&D Sys-

ems Inc.) for human cells or with goat anti-rat troponin Tntibody for rat cells. After 1 h of incubation at roomemperature, culture slides were washed and then incubatedith a phycoerythrin-conjugated secondary antibody. Celluclei were stained with 4’-6-diamidino-2-phenylindole.ardiomyogenic differentiation was quantified by countingositively stained cells.n vitro angiogenesis assay. Angiogenic potency was as-ayed by tube formation using a kit (Chemicon Interna-ional Inc., Temecula, California), according to the manu-acturer’s instructions. Briefly, cells were seeded on

Abbreviationsand Acronyms

�-SA � �-sarcomeric actin

AD-MSCs � adiposetissue–derivedmesenchymal stem cells

bFGF � basic fibroblastgrowth factor

BM-MNCs � bone marrowmononuclear cells

BM-MSCs � bone marrow–derived mesenchymal stemcells

CDCs � cardiosphere-derived cells

ELISA � enzyme-linkedimmunoadsorbent assay

HGF � hepatocyte growthfactor

HNA � human nuclearantigen

IGF � insulin-like growthfactor

IMDM � Iscove’s modifiedDulbecco’s medium

LV � left ventricular

LVEF � left ventricularejection fraction

PCR � polymerase chainreaction

SCID � severe combinedimmunodeficiency

SDF � stromal cell–derivedfactor

TUNEL � terminal deoxy-nucleotidyl transferase dUTPnick end labeling

VEGF � vascularendothelial growth factor

CMatrix-coated 96-well plates at a
density of 2 � 105 cells

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944 Li et al. JACC Vol. 59, No. 10, 2012Myocardial Repair by Different Types of Stem Cells March 6, 2012:942–53

(BM-MNCs) or 2 � 104 cells (all other cell types) per well.uman umbilical vein endothelial cells were included as

ositive controls. After 6 h, tube formation was imaged.he total tube length was then measured by using Image-ro Plus software version 5.1.2 (Media Cybernetics, Inc.,arlsbad, California).erminal deoxynucleotidyl transferase dUTP nick end

abeling assay. To quantify the resistance to oxidativetress in vitro, cells were seeded on fibronectin-coated,-chamber culture slides. After 24 h of culture, cells wereultured with or without the addition of 100 �M H2O2 to

the medium for another 24 h. Cells were fixed, andapoptotic cells were detected by terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay usingthe In Situ Cell Death Detection Kit (Roche Diagnostics,Mannheim, Germany) according to the manufacturer’sinstructions. Cell nuclei were stained with 4’-6-diamidino-2-phenylindole; apoptotic cells were counted according toTUNEL-positive nuclei.Myocardial infarction model and cell implantation.Acute myocardial infarction was created in male SCID-beige mice (10 to 12 weeks old), as described previously(6,8). Briefly, just after ligation of the left anterior descend-ing artery with 9-0 prolene, hearts were injected at 4 pointsin the infarct border zone with a total of 40 �l of one of theollowing interventions: phosphate-buffered saline (control,� 8), 1 � 105 CDCs (n � 20), 1 �105 BM-MSCs (n �

0), 1 � 105 AD-MSCs (n � 20), 1 � 106 BM-MNCshigh BM-MNCs, n � 11), or 1 � 105 BM-MNCs (lowM-MNCs, n � 9). We studied 2 dosages in BM-MNCs,

ncluding one with 10-fold more cells than in the compar-tor groups, because MNCs are smaller than the other cellypes (19) and we did not want to bias against them in termsf total transplanted cell mass.To compare the c-kit� stem cell subpopulation purified

from CDCs with the unsorted CDCs, we performed aseparate study by injecting 1 � 105 purified c-kit� cells(c-kit�, n � 16) and 1 � 105 unsorted CDCs (unsorted,n � 11) into the infarcted hearts of SCID mice, using themethods described earlier.Echocardiography. Mice underwent echocardiography 3 h(baseline) and 3 weeks after surgery by using the Vevo 770Imaging System (VisualSonics, Toronto, Ontario, Canada)(8). After the induction of light general anesthesia, thehearts were imaged 2-dimensionally in long-axis views atthe level of the greatest left ventricular (LV) diameter. LVend-diastolic volume, LV end-systolic volume, and LVejection fraction (LVEF) were measured with VisualSonicsversion 1.3.8 software from 2-dimensional long-axis viewstaken through the infarcted area. Blinded reading of echo-cardiography was conducted independently by 2 experi-enced echocardiographers (K.M. and J.T.). The resultscorrelated well (R2 � 0.66 and 0.73 for baseline and 3weeks’ measurement, respectively) (Online Fig. 1). ABland-Altman plot indicated the limit of agreement to be
from –14.07 to 13.56 (30). Thus, the averages of the 2 v
readings for LVEF in each mouse were used for statisticalanalysis.Histology. Mice were sacrificed 3 weeks after treatment.Hearts were sectioned in 5-�m sections and fixed with 4%paraformaldehyde. The engraftment of implanted humancells was identified by immunostaining for human nuclearantigen (HNA; Chemicon International Inc.). To measurecell engraftment, 10 images of the infarct and border zoneswere selected randomly from each animal. To quantify theapoptotic cells in the heart, slides were fixed and apoptoticcells were detected by TUNEL assay as described earlier.The differentiation of implanted human cells into car-diomyocytes in the infarcted hearts of SCID mice wasidentified by immunostaining with monoclonal antibodiesagainst human-specific �-sarcomeric actin (�-SA) (Sigma-

ldrich), as described previously (8,31). For morphometricnalysis, animals in each group were euthanized at 3 weeksafter cardiac function assessment), and the hearts werearvested and frozen in optimal cutting temperature com-ound. Sections every 100 �m (5-�m thick) were prepared.asson’s trichrome staining was performed per manufac-

urer’s instructions (HT15 Trichrome Stain [Masson] Kit;igma). Images were acquired with a PathScan Enabler IVlide scanner (Advanced Imaging Concepts, Princeton,ew Jersey). From the Masson’s trichrome-stained images,orphometric parameters (including infarct wall thickness

nd infarct perimeter) were measured in each section withIH ImageJ software (8,31).uantification of engraftment by real-time polymerase

hain reaction. Quantitative polymerase chain reactionPCR) was performed 3 weeks after cell injection touantify cell engraftment. Male human cells were injectedo enable detection of the SRY gene located on the Yhromosome as a marker of engrafted cells (31). The wholeouse heart was harvested, weighed, and homogenized.he TaqMan assay was used to quantify the number of

ransplanted cells with the human SRY gene as templateApplied Biosystems, Foster City, California). A standardurve was generated with multiple dilutions of genomicNA isolated from the injected CDCs to quantify the

bsolute gene copy numbers. All samples were spiked withqual amounts of genomic DNA from noninjected mouseearts as a control. For each reaction, 50 ng of genomicNA was used. Real-time PCR was performed in triplicateith an Applied Biosystems 7900 HT Fast real-time PCRachine. The number of engrafted cells per heart was

uantified from the standard curve.tatistical analysis. Statistical analysis was performed in-ependently (by H.K.). All results are presented as mean �D except as noted. Data sets were first tested for normalityKolmogorov-Smirnov test) and variance (Levene’s test). Ifoth were assured, statistical significance was determined by-way analysis of variance followed by post hoc test (SPSSI, SPSS Inc., Chicago, Illinois). If either normality or
ariance tests failed, nonparametric tests (Kruskal-Wallis

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test followed by Dunn’s post-test) were used. Differenceswere considered statistically significant when p � 0.05.

Results

Characterization of cell phenotypes. Unlike BM-MNCs,which grow in suspension as small round cells, all other celltypes studied (CDCs, BM-MSCs, and AD-MSCs) typi-cally grow as adherent monolayers (Fig. 1A). Flow cytom-etry distinguished BM-MNCs from other cell types by thepredominant expression of pan-hematopoietic markerCD45 (74.7%), compared with �1% in CDCs, BM-MSCs,and AD-MSCs (Fig. 1B). Conversely, �99% of CDCs,BM-MSCs, and AD-MSCs expressed CD105, a trans-forming growth factor–beta receptor subunit commonlyassociated with MSCs. However, these 3 cell types can bedistinguished by CD90: �99% of BM-MSCs and 85% of

D-MSCs expressed CD90 but only 18% of CDCs did so.D90 (well known as Thy-1) was originally discovered as a

hymocyte antigen (32). In humans, Thy-1 is also expressedy endothelial cells, smooth muscle cells, a subset ofD34� bone marrow cells, and umbilical cord blood–,broblast-, and fetal liver–derived hematopoietic cells.D90 is widely used as a marker of a variety of stem cells

e.g., MSCs, hepatic stem cells, keratinocyte stem cells,utative endometrial progenitor/stem cells, hematopoietictem cells) (33,34). On the basis of these findings, CDCseem to contain a minority of fibroblast and/or weaklyommitted hematopoietic cells, whereas such populationsominate in the cells of bone marrow and adipose origins.

Figure 1 Morphology and Phenotype Characterization

(A) Representative images of cardiosphere-derived cells (CDCs), bone marrow–decells (AD-MSCs), and bone marrow–derived mononuclear cells (BM-MNCs) after 3growth and stromal (mesenchymal) cell-like morphology; BM-MNCs have smaller sCD117 (c-kit), and CD133 in CDCs, BM-MSCs, AD-MSCs, and BM-MNCs. Bar � 50

Alternatively, the CD90 in CDCs may simply mark thecardiac mesenchymal subpopulation.In vitro secretion of growth factors. Increasing evidencesupports the generalization that cell therapy boosts cardiacfunction largely via paracrine mechanisms (25). We thuscompared the production of 6 growth factors (angiopoietin-2,bFGF, HGF, IGF-1, SDF-1, and VEGF) by the variouscell types. CDCs were unique in their ability to secrete largeamounts of all growth factors (Fig. 2A). In contrast, theother cell types failed to express meaningful levels of 1 ormore growth factors: BM-MNCs produced little VEGFand SDF-1; BM-MSCs secreted little IGF-1 and bFGF;and AD-MSCs were not rich sources of HGF and SDF-1.Figure 2B depicts schematically the secretion of each of the6 cytokines in each given cell type, as wheel-and-spokediagrams in which the length of each spoke is propor-tional to the growth factor concentration in conditionedmedia. The symmetrical starburst pattern highlights therelatively high and distributed paracrine factor produc-tion of CDCs.

One limitation of our comparison is that we used 10-foldhigher concentrations of BM-MNCs in these experiments,with the rationale that these cells are much smaller than theother 3 cell types (24) and thus have a lower secretorybiomass. Another limitation of our comparison is the factthat the various human cell types originated from differentsubjects. To ensure that the findings did not simply reflectdonor-specific idiosyncrasies, we compared growth factorsecretion by the various cell types derived from individual

esenchymal stem cells (BM-MSCs), adipose tissue–derived mesenchymal stemn culture under 20% O2. CDCs, BM-MSCs, and AD-MSCs demonstrated adherent

round shape. (B) Expression profile of CD29, CD31, CD34, CD45, CD90,

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rats. In agreement with the findings in human stem cells,higher levels of HGF, IGF-1, and VEGF were alsoobserved in media conditioned by rat CDCs than by ratBM-MSCs, AD-MSCs, and BM-MNCs, all collectedfrom the same animals (Online Fig. 2). The well-balancedrelease of growth factors in vitro confirms the notion thatCDCs are particularly rich biological factories (25,35), afeature that may favor enhanced myocardial repair throughparacrine mechanisms after implantation into the heart.Cardiomyogenic differentiation. Another potential ad-vantage of CDCs is their reported ability to differentiateinto cardiomyocytes (8,24,25,31), although this propensityhas not been compared quantitatively with other cell types.We therefore examined the ability to undergo spontaneouscardiomyogenic differentiation in vitro by immunostainingfor cardiac-specific troponin T. Many human CDCs ex-pressed troponin T (Fig. 3A), in contrast to human BM-MSCs, AD-MSCs, or BM-MNCs, few of which werepositive for troponin T. Quantitative analysis revealed thatapproximately 9% of CDCs expressed troponin T, whereas

Figure 2 Comparison of In Vitro Production of Growth Factors F

(A) Concentrations of angiopoietin-2, basic fibroblast growth factor (bFGF), hepato(SDF)-1, and vascular endothelial growth factor (VEGF) measured by using enzymesecretion of each of the 6 cytokines in each given cell type, as wheel-and-spoke dtration in conditioned media and is normalized to that from CDCs. The symmetricaAbbreviations as in Figure 1.

�1% did so in the other cell types (Fig. 3B). Similar w

findings were observed using rat CDCs, BM-MSCs, AD-MSCs, and BM-MNCs, all collected from the same ani-mals (Online Fig. 3).Tube formation. We quantified the angiogenic ability ofthe various cell types using an in vitro tube-forming assay(36). All cell types could form capillary-like networks onMatrigel (BD Biosciences, Bedford, Massachusetts) within6 h, with the exception of BM-MNCs (Fig. 3C). Quanti-tative analysis revealed that the mean tube length of thecapillary-like networks was greater in CDCs than in theother cell types (p � 0.05) (Fig. 3D). Thus, CDCs, at leasty this simple in vitro assay, are superior in mediating theormation of capillary-like structures.

esistance to oxidative stress. Enhanced cell resilience toxidative stress favors transplanted cell engraftment andunctional benefit (35). We assayed sensitivity to oxidativetress by exposing cells to H2O2, a powerful oxidant. After4 h of exposure to 100 �M of H2O2, the number ofpoptotic cells tended to be lower in human CDCs than inuman BM-MNCs (p � 0.067) (Online Fig. 4), but there

Cultured Cells

owth factor (HGF), insulin-like growth factor (IGF)-1, stromal cell–derived factorimmunoadsorbent assay (n � 3) are depicted. (B) Schematic depicting thes in which the length of each spoke is proportional to the growth factor concen-urst pattern highlights the uniquely well-balanced paracrine profile of CDCs.

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and AD-MSCs. In rat cells, higher apoptosis was observedin BM-MNCs compared with any of the other 3 cell typesafter H2O2 exposure (p � 0.05) (Online Fig. 5). Takentogether, these data highlight a relative deficiency of BM-MNCs in terms of resistance to oxidative stress.Cell engraftment and in vivo differentiation. Althoughmost of the benefit of cell therapy is now recognized to beindirect, study after study has revealed a positive correlationbetween long-term cell engraftment and functional benefit(10,19–22,25). We therefore evaluated the engraftment anddifferentiation of human cells 3 weeks after direct intramyo-cardial injection into the infarcted hearts of SCID mice.Consistent with previous reports (7,8,23–25,31), histologyresults revealed expression of �-SA in some of the survivingprogeny of human CDCs (positive for HNA) (Fig. 4A),confirming the cardiomyogenic differentiation in vivo. Incontrast, human cells positive for �-SA were observed rarelyand inconsistently in mice injected with BM-MSCs, AD-MSCs, and BM-MNCs (data not shown). Quantitativeimage analysis confirmed that the engraftment (i.e., thenumbers of HNA�cells) was greater in mice implanted withhuman CDCs than with comparator cells (p � 0.05) (Fig.4B). In addition, the numbers of cardiomyocytes derivedfrom the transplanted cells (HNA�/�-SA�) were greater in

Figure 3 In vitro Myogenic Differentiation and Angiogenesis As

(A) Troponin T, with distinct myocyte-like appearance, was expressed spontaneousexpressed in BM-MSCs, AD-MSCs, and BM-MNCs. (B) Quantitative analysis of troptive), and AD-MSCs and BM-MNCs (approximately 0.1% positive). (C) CDCs, BM-MMNCs did not form similar structures under these conditions. (D) Quantitation andAbbreviations as in Figure 1.

ice implanted with human CDCs than with any of the m

ther cell types (p � 0.01) (Fig. 4C). Quantitative PCRonfirmed the histological analysis: more human cells wereetected in the CDC group than in the low BM-MNCroup (p � 0.05) (Fig. 4D) 3 weeks after injection. Also,ccording to PCR analysis, there was a trend indicatingigher engraftment in the CDC group than in the BM-SC and AD-MSC groups.

ell apoptosis. In addition to tissue regeneration, tissuereservation may be a salutary component of cell therapy forcute myocardial infarction (25). To evaluate this possibil-ty, we quantified apoptotic nuclei in infarcts of control micend mice injected with each of the comparator cell types.UNEL staining revealed apoptotic nuclei in the infarctedearts 3 weeks after treatment (Figs. 5A and 5B). Weounted the total number of apoptotic cells in the infarctnd peri-infarct area. The hearts of mice implanted withDCs exhibited fewer TUNEL-positive cells, comparedith all other cell-treated groups (p � 0.05) (Fig. 5C). We

peculate that this enhanced tissue preservation may be dueo the larger amount of pro-angiogenic and antiapoptoticactors produced by CDCs (19,25,35).

ardiac function. The most meaningful indicator of cellotency, in practice, is the ability to produce functionalenefit after transplantation into the injured heart. We

fraction of CDCs cultured for 7 days. This cardiac-specific marker was rarelyexpression (n � 3) in CDCs (9% of the cells positive), BM-MSCs (0.4% posi-

nd AD-MSCs produced capillary-like tube formations in extracellular matrix. BM-rison of tube formation capacity by the different cell types (n � 3). Bars � 50 �m.

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all images were interpreted blindly and independently by 2experienced echocardiographers. Figure 6 summarizes theresults. The LVEF at baseline (i.e., 2 h post-infarction) wascomparable, indicating similar ischemic injury amonggroups. Among the various treatments, the implantation ofCDCs resulted in the greatest LEVF at 3 weeks, and it isthe only group that was significantly better than the controls(p � 0.05 vs. control). The other cell types, although higheron average than controls, had no clear functional benefitstatistically. Echocardiography also revealed smaller end-diastolic and end-systolic LV volumes in the CDC-treatedanimals (Online Fig. 6).Ventricular remodeling. Potentiating the functional ben-efits of cell therapy is attenuation of adverse ventricularremodeling (8). To evaluate this process, we examined themorphological consequences of transplantation of the vari-ous cell types on myocardial infarct size and wall thinning.Heart morphometry at 3 weeks revealed severe LV chamberdilation and infarct wall thinning in the control hearts (Fig. 7A,Online Fig. 7). In contrast, all the cell-treated groupsexhibited attenuated LV remodeling. Compared with con-trols, the implantation of any type of human cells decreasedfractional infarct perimeter and, conversely, increased the

Figure 4 Cell Engraftment and In Vivo Myogenic Differentiation

(A) Immunostaining shows some human CDCs (green; human nuclear antigen [HNafter implantation into infarcted mice hearts. (B) Quantitation of engraftment (HNAcells (HNA�/�-SA�cells). Bar � 20 um. (D) Quantitation of engraftment by usi4’,6-diamidino-2-phenylindole; other abbreviations as in Figure 1.

minimal infarct wall thickness 3 weeks after treatment (p � p

0.05 vs. control group). The protective effect was greatest inthe CDC-treated hearts, which had thicker infarcted walls(p � 0.01) (Fig. 7B) but a smaller fractional infarct

erimeter (p � 0.05) (Fig. 7C) than any of the otherell-treated groups.nsorted CDCs versus the c-kit�–purified cell subpopulation.

Having established that CDCs are the most effective celltype among those studied, we moved on to consider howCDCs compare with purified c-kit� cells. This comparisonis germane because c-kit has been argued to identify cardiacstem/progenitor cells (1,7–9), and a purified c-kit� cardiac-erived cell product is currently being tested clinically. Therefore,n a separate study, we compared unsorted CDCs with equalumbers of c-kit� stem cells purified from CDCs by usingagnetic cell sorting. Purified c-kit� cells were inferior to

nsorted CDCs in terms of functional benefit after trans-lantation into the infarcted heart, although they did outper-orm vehicle-injected controls (Fig. 8A). The sorting proceduretself did not compromise cell functional efficacy, as CDCsorted for CD105 (expressed by �99% of CDCs) exhibitedn LVEF comparable to that of unsorted CDCs (data nothown). To investigate one potential mechanism for theunctional superiority of CDCs, we measured a variety of

pressing �-sarcomeric actin (�-SA), indicating myogenic differentiation, 3 weeksls). (C) Quantitation of cardiomyocytes differentiated from transplanted humanntitative polymerase chain reaction (PCR) (n � 3 mice per group). DAPI �

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unsorted cells. Indeed, unsorted CDCs produced higheramounts of paracrine factors in vitro compared with purifiedc-kit� cells (Fig. 8B), providing one potential rationale forheir enhanced benefit.

iscussion

y head-to-head direct comparison, we demonstrated aunctional superiority of heart-derived cells compared with

types of adult stem cells of extracardiac origin: BM-NCs, and MSCs from bone marrow or adipose tissue.

urthermore, the superiority of CDCs for myocardial repairas consistent with their well-balanced secretion of para-

Figure 5 Cell Apoptosis

Representative images of terminal deoxynucleotidyl transferase dUTP nick end labwith (A) CDCs and (B) phosphate-buffered saline. (C) Quantitative assessment ofcontrol, is shown (n � 3 mice per group). Bar � 500 um. Abbreviations as in Figu

Figure 6 Cardiac Function

Left ventricular ejection fraction (LVEF) at baseline (4 h post–myocardial infarc-tion) did not differ among groups, indicating a similar infarct size in animals ofall groups. After 3 weeks, LVEF was higher in mice implanted with CDCs, com-pared with controls injected with saline only. Differences between any other 2groups were not significant. Data are presented as mean � SEM. Abbrevia-tions as in Figure 1.

rine factors as well as their higher cardiomyogenic differ-ntiation capacity and engraftment, although further in vivontervention experiments (e.g., with blocking antibodies)ill be required to identify the precise mechanisms of

unctional superiority.We selected 4 cell types for comparison that have been

sed clinically and are considered among the most promis-ng at present for myocardial repair/regeneration. Previoustudies (19–22) have shown that the implantation of BM-

NCs, BM-MSCs, and AD-MSCs into the damagedeart can improve cardiac function, likely through paracrineechanisms, with rare events of myogenic differentiation.espite the paucity of direct regeneration of new myocar-

ium from these stem cells of extracardiac origin, themprovement of cardiac function reported in previous ran-omized clinical trials is encouraging (10–18).One distinctive feature of resident cardiac stem cells is their

bility to undergo consistent cardiomyogenic and angiogenicifferentiation (1–9,23–25), a finding confirmed here. Al-hough almost 10% of CDCs expressed troponin T in vitro,uman CDCs expressing �-SA were infrequently observed inhe infarcted hearts of SCID mice 3 weeks after treatment.urthermore, we did not examine whether these �-SA–

positive human CDCs were physiologically integratedwith the resident cardiomyocytes of mice. Therefore,regeneration of new functional myocardium directly fromthe implanted human CDCs is a consistent finding butone of questionable significance. The present evidencesupports the notion that mechanism of functional benefitafter CDC implantation, as with other cell types, ispredominantly dependent on paracrine effects and re-cruitment of endogenous regeneration (25).

We thus compared the secretion of growth factors that

TUNEL)-positive cells in the infarcted hearts of mice 3 weeks after cell treatmentL-positive cells in the myocardium of mice treated with different cell types andand 4.

eling (TUNE

res 1

generally are recognized to play critical roles in angiogene-


sis, antiapoptosis, or cardioprotection. Platelet-derivedgrowth factor was undetectable in media conditioned by anyof the 4 cell types, but, beyond even our own expectations,CDCs robustly produced a variety of growth factors, in-cluding angiopoietin, bFGF, HGF, IGF-1, SDF-1, andVEGF. Although BM-MSCs, AD-MSCs, and BM-MNCs could produce some of these factors in levelscomparable to CDCs, the paracrine profile was uniquelywell balanced in CDCs. The robust production of diverseparacrine factors by CDCs provides a potential mechanismunderlying their superiority for functional myocardial repair,but further research will be required to understand theprecise role of each paracrine factor, as well as the roles ofothers known to be secreted but not quantified here (37).The use of the same medium (IMDM) for all cell typesavoids possible confounding effects due to different cultureconditions; nevertheless, the fact that cells were cultureduniformly may minimize favorable features unique to eachcell type that might be more apparent with media optimizedfor each cell type.

Tube formation assays are commonly used to measure theangiogenic potential of endothelial cells (36). The superi-

Figure 7 Ventricular Remodeling

(A) Representative images of Masson’s staining of infarcted mice hearts after impses (n � 3 mice per group) of (B) left ventricular wall thickness and (C) infarct pecompared with BM-MSCs, AD-MSCs, and BM-MNC treatment, although implantatiocontrol treatment with saline injection only. Abbreviations as in Figure 1.

ority of tube formation observed in CDCs indicates their

angiogenic potential, which is in agreement with CDCs’robust secretion of pro-angiogenic cytokines (Fig. 2). Theobservation that BM-MNCs failed to form any tubes maybe due to the fact that MNCs need a longer time (24 h) toform tubes (36), whereas we performed the assay at the 6-htime point. Because we did not stain for endothelial markers(e.g., CD31), it is unknown whether these tube structuresactually represent endothelial cells differentiated from thevarious cell types. Indeed, it has been reported that mesen-chymal and fibroblast cells assist in blood vessel formationby aligning into tube networks (38).

We also found CDCs were more resistant to oxidativestress-induced apoptosis in vitro (Online Figs. 4 and 5) andMI-induced apoptosis in vivo (Fig. 5). The superiority ofCDCs to other cell types in enhancing host tissue preser-vation is striking. Therefore, it is conceivable that this actionis responsible for the superior impact of CDCs in terms ofpreserving cardiac function and attenuating pathologicalremodeling, although the relative importance of the variousfavorable effects remains to be defined. It should be notedthat we assayed apoptosis in vivo 3 weeks after myocardialinfarction/cell treatment. At that time point, the acute

ion of different types of human cells or saline injection only. Quantitative analy-r show that remodeling was attenuated more efficiently by CDC implantation,M-MSCs, AD-MSCs, and BM-MNCs resulted in less remodeling compared with

lantatrimeten of B

phase of cell death due to ischemia might have already

beesa

bbresirec

nfl

C

Idltdam


resolved; therefore, the apoptosis we observed was a reflec-tion of long-term remodeling and heart failure. One limi-tation of this experiment is that we did not perform doublestaining for TUNEL- and HNA-positive nuclei. Therefore,we cannot distinguish whether apoptosis is reduced inendogenous cells, transplanted cells, or both. In addition,our results do not exclude the possibility that different levelsof inflammatory infiltration among groups might contributeto observed differences in apoptotic rates. Nevertheless, thesuperior tissue preservation capacity of CDCs is consistentwith their robust secretion of antiapoptotic and pro-angiogenic factors.

We did not include a number of other stem cell types inour comparison, including myoblasts and peripheral blood–derived endothelial progenitors, although they have alreadybeen tested in clinical trials (17,18). The major reason is therelative lack of enthusiasm in the field for these cell types ascandidates for further clinical development for cardiacregeneration (although endothelial progenitors do havepositive effects on angina and critical limb ischemia [17]).We also did not select embryonic stem cell- or inducedpluripotent stem cell-derived cardiomyocytes for compari-son because they are far from clinical applicability at present,although both may excel in the direct production of newfunctional myocardium (39,40).

We did, however, compare the therapeutic potency ofcardiac progenitor cells purified by virtue of their c-kit-positivity (35,41), relative to unselected CDCs (42). Weconfirmed the reported ability of c-kit� cardiac cells tooost cardiac function post-infarction (1,9), but we discov-red that c-kit� cells are not as potent as the CDC mixtureither in terms of functional benefit or in paracrine factorecretion. One trivial explanation for this finding would be

Figure 8 Comparison of Purified c-kit� Stem Cells and Unsorte

(A) 3 weeks after infarction, left ventricular ejection fraction (LVEF) was higher in mCDCs, although these purified c-kit� stem cells also improved cardiac function whcells was used for culture, the purified c-kit� stem cells released fewer VEGF, SDF

ntibody-related interference with cell potency, but we

elieve that is unlikely. The c-kit antibody used (43) haseen shown to interfere minimally with ligand binding,eceptor phosphorylation, and internalization in c-kit–xpressing cell lines (44). Also, magnetic-activated cellorting for mast cells using this c-kit antibody neithernduced histamine release nor impaired the ability of cells toelease histamine when stimulated (45). We must acknowl-dge, however, that subtle differences in specific sorting andulture methods for c-kit� cells may affect the efficacy of the

cells. Nevertheless, the finding that the natural CDCmixture is superior to the purified c-kit� subpopulation addsa new dimension to the emerging notion that mesenchymalcells favor endogenous cardiac regeneration (10,46), at leastpartly via the attraction of c-kit� cells. We conjecture thatthe stromal and mesenchymal cells synergize with the c-kit�

cells in the natural CDC mixture to enhance overallparacrine potency and thereby boost functional benefit;alternatively, c-kit� cells may be tangential to the mecha-

ism of benefit of CDCs. More experiments are required toesh out these ideas.

onclusions

n this first comprehensive head-to-head comparison of 4ifferent cell types in the same animal model in the same

aboratory, with blinded analysis, CDCs were superior inerms of paracrine factor secretion, angiogenesis, car-iomyogenic differentiation, ischemic tissue preservation,ntiremodeling effects, and functional benefit. The CDCixture was more potent than the c-kit� subpopulation.

Ongoing and future clinical studies will serve as the ultimatetest of potency, but the present results give good reason for

Cs

at received unsorted CDCs than those with c-kit� cells purified from the samepared with controls injected with saline only. (B) Although the same number of, and HGF than the unsorted CDCs. Abbreviations as in Figures 1 and 2.

d CD

ice then com, IGF-1

optimism regarding the therapeutic potential of CDCs.

1

1

1

1

1

1

1

1

1

1

2

2

2

2

2

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2

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NOTE: A report has appeared of the first clinical trialusing cardiosphere-derived cells: Makkar RR, Smith RR,Cheng K, et al. Intracoronary cardiosphere-derived cells forheart regeneration after myocardial infarction (CADU-CEUS): a prospective, randomised phase 1 trial. Lancet2012 Feb 14 [E-pub ahead of print]; doi:10.1016/S0140-6736(12)60195-0.

Reprint requests and correspondence: Dr. Eduardo Marban,Cedars-Sinai Heart Institute, 8700 Beverly Boulevard, Los Ange-les, California 90048. E-mail: [email protected].

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Key Words: cardiac stem cells y mesenchymal stem cells y myocardialregeneration y paracrine effects.

APPENDIX

For supplemental figures on the study results,
please see the online version of this article.

direct comparison of different stem cell types and subpopulations

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